Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by the expressions of involuntary movements (chorea), dementia, and psychiatric disorders (J. Med. 315, 1267-1276, 1986). The disease generally develops during the middle ages of 30-50 years old, however some of the cases may develop very earlier or later in life than the ages. The symptom is progressive, and most of the cases result in death in 10-20 years after the development of the secondary complication of the involuntary movements. By the examination of individual brains after the death resulting from Huntington's disease, a selective loss of neuronal cells was proved to affect to the striatum. A huntingtin gene, which is the causative gene for Huntington's disease, is mapped in a region of 2.2 Mb located between loci D4S126 and D4S98 in cellular genetic sub-band at the end of the short arm of chromosome 4 in human (Neuron 3, 183-190, 1989, J. Hum. Genet. 49, 7-16, 1991, Am. J. Hum. Genet. 51, 357-362, 1992).
Huntington's disease is a genetic neurodegenerative disorder resulted from progressively losing brain striatum neuronal cells after CAG repeats expanded in exon 1 of huntingtin gene transcription and translated into polyglutamine (poly Q) tract (Annu. Rev. Med. 47, 201-209, 1996) In other words, Huntington's disease is caused by abnormally expanded CAG repeats on the exon 1 portion of huntingtin gene and results in the selective loss of brain striatal nerves. The huntingtin gene codes for cytoplasmic protein of molecular weight 348 kDa called huntingtin, widely expresses in both central nervous system (CNS) and non-central nervous system (non-CNS) tissues. The CGA triplet sequence (CAG triplets) of HD gene is translated into polyglutamine (poly Q) in huntingtin protein. Typically, normal and mutant huntingtin alleles carry 6 to 37 and 35 to 180 CAG repeats, respectively.
In recent years, as a method for the treatment of Huntington's disease, methods of treating huntingtin genes, targeting huntingtin genes, using antagonistic substances against huntingtin protein expressing huntingtin genes, and the like, have been disclosed. For example, Publication of Japanese Laid-Open Patent Application No. 1995-67661 discloses the treatment methods of: substituting mutant huntingtin genes for normal genes after inserting DNAs expressing normal huntingtin proteins into cells of patients; introducing genes that encode sequences capable of transcribing and expressing antisense RNAs of huntingtin genes of Huntington's disease, into cells of patients; administering antagonists to huntingtin proteins of Huntington's disease; or the like. As a treatment method of autosomal dominant disorders such as Huntington's disease, the treatment method by the allele-specific targeting that targets against RNAs of Huntington's disease, has been also disclosed in Published Japanese translation of PCT international publication No. 2003-503008. However, when considered from the viewpoints of the complication and stability of introducing genes, or the treatment effect obtained, these treatment methods have not always worked out as expected.
On the other hand, in some kind of creature (Caenorhabditis elegans), it has been recently found that the gene expression can be specifically inhibited by double-stranded RNAs (Nature 391, 806-811, 1998, WO99/32619). This phenomenon is that double-stranded RNAs (dsRNAs) composed of sense- and antisense-strand RNAs, which are homologous to certain genes, destroy the homologous part in the transcription products (mRNAs) of the genes, and called RNAi (RNA interference). Later the phenomenon was found in lower eukaryotic cells including various kinds of animals (Cell 95, 1017-1026, 1998, Proc. Natl. Acad. Sci. USA 95, 14687-14692, 1998, Proc. Natl. Acad. Sci. USA 96, 5049-5054, 1999) and plants (Proc. Natl. Acad. Sci. USA 95, 13959-13964, 1998).
In the early days of the discovery, RNAi was believed to be difficult to use in mammalian cells, since apoptosis was induced by underlying immune function in cells and the cells died, when around 30 or more bp of dsRNAs were introduced into the cells. However, RNAi was also identified to occur in mouse early embryos and in mammalian cultured cells in 2000, and it has become apparent that the RNAi induction mechanism itself also exists in mammalian cells (FEBS Lett 479, 79-82, 2000, WO01/36646).
If the expression of certain genes or gene clusters is inhibited in mammals by using such an RNAi function, it would be apparently useful. Since many of the diseases (such as cancer, endocrine disease, and immunological disease) are developed by abnormal expression of certain genes or gene clusters in mammals, the inhibition of the genes or gene clusters may be used to treat these symptoms. In addition, diseases may be developed due to the expression of mutant protein, in these cases, the diseases can be treated by suppressing the mutant allele expression. Furthermore, such gene-specific inhibition can be used for the treatment of viral disease that was caused by, for example, retrovirus (viral genes in retrovirus are incorporated into their host genome and expressed) such as HIV.
The dsRNAs inducing the function of RNAi were initially considered to require the introduction of around 30 or more bp of dsRNAs into the cells, however, it has recently become apparent that the shorter (21-23 bp) dsRNAs (short double-stranded RNAs: siRNA; small interfering RNA) can induce RNAi without exhibiting cytotoxicity even in mammalian cell system (Nature 411, 494-498, 2001). The siRNA is recognized as a powerful tool to suppress gene expression at all the developmental stages of somatic cells, and can be expected as a method to suppress disease-causing gene expression before the development of the disease in progressive genetic diseases and the like. But it has not been reported yet that a method to suppress gene-specific expression by using such dsRNAs, effectively applied for the genetic disease of Huntington's disease (HD).
The subject of the present invention is to provide the double-stranded RNAs (siRNAs) composed of sense- and antisense-strand RNAs homologous to the certain sequences targeted by huntingtin mRNAs which can suppress the expression of huntingtin gene, the huntingtin gene expression inhibitors composed of the double-stranded RNAs, the preventives and/or the remedies for Huntington's disease containing the expression inhibitor as an active ingredient, and the like.
Huntington's disease is a genetic neurodegenerative disorder resulted from progressively losing brain striatal neuronal cells after CAG repeats expanded in exon 1 of huntingtin gene transcription and translated into polyglutamine (poly Q) tract. When the huntingtin mRNAs at upstream of CAG repeats were examined, the present inventors found two sites containing the specific sequences which are effective targets of siRNAs. Consequently, as the dsRNA sequences homologous to these sequences; a) siRNA-5′UTR targeting 5′-untransrated region, and; b) siRNA-HDexon 1 targeting a region at immediately upstream of CAG repeats, furthermore; c) as currently known, for the only difference between normal and mutant huntingtin genes is the lengths of the CAG repeats, three siRNAs of siRNA-CAG were made directly targeting the CAG repeats, after analyzing the effect of the siRNAs by using tissue culture models or Huntington's disease mouse models, it was found that the siRNA-HDexon 1 quite efficiently suppressed the huntingtin gene expression and the development of Huntington's disease, then the present invention was completed.